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Abstract Background Long-read sequencing is increasingly used to uncover structural variants in the human genome, both functionally neutral and deleterious. Structural variants occur more frequently in regions with a high homology or repetitive segments, and one rearrangement may predispose to additional events. Bartter syndrome type 3 (BS 3) is a monogenic tubulopathy caused by deleterious variants in the chloride channel gene CLCNKB, a high proportion of these being large gene deletions. Multiplex ligation-dependent probe amplification, the current diagnostic gold standard for this type of mutation, will indicate a simple homozygous gene deletion in biallelic deletion carriers. However, since the phenotypic spectrum of BS 3 is broad even among biallelic deletion carriers, we undertook a more detailed analysis of precise breakpoint regions and genomic structure. Methods Structural variants in 32 BS 3 patients from 29 families and one BS4b patient with CLCNKB deletions were investigated using long-read and synthetic long-read sequencing, as well as targeted long-read sequencing approaches. Results We report a ~3 kb duplication of 3′-UTR CLCNKB material transposed to the corresponding locus of the neighbouring CLCNKA gene, also found on ~50 % of alleles in healthy control individuals. This previously unknown common haplotype is significantly enriched in our cohort of patients with CLCNKB deletions (45 of 51 alleles with haplotype information, 2.2 kb and 3.0 kb transposition taken together, p=9.16×10−9). Breakpoint coordinates for the CLCNKB deletion were identifiable in 28 patients, with three being compound heterozygous. In total, eight different alleles were found, one of them a complex rearrangement with three breakpoint regions. Two patients had different CLCNKA/CLCNKB hybrid genes encoding a predicted CLCNKA/CLCNKB hybrid protein with likely residual function. Conclusions The presence of multiple different deletion alleles in our cohort suggests that large CLCNKB gene deletions originated from many independently recurring genomic events clustered in a few hot spots. The uncovered associated sequence transposition haplotype apparently predisposes to these additional events. The spectrum of CLCNKB deletion alleles is broader than expected and likely still incomplete, but represents an obvious candidate for future genotype/phenotype association studies. We suggest a sensitive and cost-efficient approach, consisting of indirect sequence capture and long-read sequencing, to analyse disease-relevant structural variant hotspots in general.

Abstract Background The term congenital ocular motor apraxia (COMA), coined by Cogan in 1952, designates the incapacity to initiate voluntary eye movements performing rapid gaze shift, so called saccades. While regarded as a nosological entity by some authors, there is growing evidence that COMA designates merely a neurological symptom with etiologic heterogeneity. In 2016, we reported an observational study in a cohort of 21 patients diagnosed as having COMA. Thorough re-evaluation of the neuroimaging features of these 21 subjects revealed a previously not recognized molar tooth sign (MTS) in 11 of them, thus leading to a diagnostic reassignment as Joubert syndrome (JBTS). Specific MRI features in two further individuals indicated a Poretti–Boltshauser syndrome (PTBHS) and a tubulinopathy. In eight patients, a more precise diagnosis was not achieved. We pursued this cohort aiming at clarification of the definite genetic basis of COMA in each patient. Results Using a candidate gene approach, molecular genetic panels or exome sequencing, we detected causative molecular genetic variants in 17 of 21 patients with COMA. In nine of those 11 subjects diagnosed with JBTS due to newly recognized MTS on neuroimaging, we found pathogenic mutations in five different genes known to be associated with JBTS, including KIAA0586, NPHP1, CC2D2A, MKS1, and TMEM67. In two individuals without MTS on MRI, pathogenic variants were detected in NPHP1 and KIAA0586, arriving at a diagnosis of JBTS type 4 and 23, respectively. Three patients carried heterozygous truncating variants in SUFU, representing the first description of a newly identified forme fruste of JBTS. The clinical diagnoses of PTBHS and tubulinopathy were confirmed by detection of causative variants in LAMA1 and TUBA1A, respectively. In one patient with normal MRI, biallelic pathogenic variants in ATM indicated variant ataxia telangiectasia. Exome sequencing failed to reveal causative genetic variants in the remaining four subjects, two of them with clear MTS on MRI. Conclusions Our findings indicate marked etiologic heterogeneity in COMA with detection of causative mutations in 81% (17/21) in our cohort and nine different genes being affected, mostly genes associated with JBTS. We provide a diagnostic algorithm for COMA.

Abstract Several high-throughput antibody-free methods for RNA modification detection from sequencing data have been developed. We present JACUSA2 as a versatile software solution and comprehensive analysis framework for RNA modification detection assays that are based on either the Illumina or Nanopore platform. Importantly, JACUSA2 can integrate information from multiple experiments, such as replicates and different conditions, and different library types, such as first- or second-strand cDNA libraries. We demonstrate its utility, showing analysis workflows for N6-methyladenosine (m6A) and pseudouridine (Ψ) detection on Illumina and Nanopore sequencing data sets. Our software and its R helper package are available as open source solutions.

Abstract Cell-free DNA (cfDNA) analysis has become essential in cancer diagnostics and prenatal testing. We present cfNOMe, a two-in-one method of measuring cfDNA cytosine methylation and nucleosome occupancy in a single assay using non-disruptive enzymatic cytosine conversion and a custom bioinformatic pipeline. We show that enzymatic cytosine conversion better preserves cfDNA fragmentation information than does bisulfite conversion. Whereas previously separate experiments were required to study either epigenetic marking, cfNOMe delivers reliable results for both, enabling more comprehensive and inexpensive epigenetic cfDNA profiling. cfNOMe has the potential to advance biomarker discovery and diagnostic usage in diseases with systemic perturbations of cfDNA composition.

Abstract Background Mismatch repair (MMR)-deficiency increases the risk of colorectal tumorigenesis. To determine whether the tumors develop on a normal or disturbed epigenetic background and how radiation affects this, we quantified genome-wide histone H3 methylation profiles in macroscopic normal intestinal tissue of young radiated and untreated MMR-deficient VCMsh2 LoxP/LoxP (Msh2 −/− ) mice months before tumor onset. Results Histone H3 methylation increases in Msh2 −/− compared to control Msh2 +/+ mice. Activating H3K4me3 and H3K36me3 histone marks frequently accumulate at genes that are H3K27me3 or H3K4me3 modified in Msh2 +/+ mice, respectively. The genes recruiting H3K36me3 enrich in gene sets associated with DNA repair, RNA processing, and ribosome biogenesis that become transcriptionally upregulated in the developing tumors. A similar epigenetic effect is present in Msh2 +/+ mice 4 weeks after a single-radiation hit, whereas radiation of Msh2 −/− mice left their histone methylation profiles almost unchanged. Conclusions MMR deficiency results in genome-wide changes in histone H3 methylation profiles preceding tumor development. Similar changes constitute a persistent epigenetic signature of radiation-induced DNA damage.

Abstract Background Serrated or Hyperplastic Polyposis Syndrome (SPS, HPS) is a yet poorly defined colorectal cancer (CRC) predisposition characterised by the occurrence of multiple and/or large serrated polyps throughout the colon. A serrated polyp-CRC sequence (serrated pathway) of CRC formation has been postulated, however, to date only few molecular signatures of serrated neoplasia (BRAF, KRAS, RNF43 mutations, CpG Island Methylation, MSI) have been described in a subset of SPS patients and neither the etiology of the syndrome nor the distinct genetic alterations during tumorigenesis have been identified. Methods To identify somatic point mutations in potential novel candidate genes of SPS-associated lesions and the involved pathways we performed exome sequencing of eleven early serrated polyps obtained from a 41 year-old female patient with clinically confirmed SPS. For data filtering and analysis, standard pipelines were used. Somatic mutations were identified by comparison with leukocyte DNA and were validated by Sanger sequencing. Results The BRAF p.V600E or KRAS p.G12D mutation was identified in six polyps (~50%) and not found in polyps from the distal colon. In addition, we found seven unique rare somatic alterations of seven different genes in four serrated tumours, all of which are missense variants. The variant in ABI3BP and CATSPERB are predicted to be deleterious. No established cancer gene or candidate genes related to serrated tumorigenesis were affected. Conclusions Somatic mutations seem to be rare events in early hyperplastic and serrated lesions of SPS patients. Neither frequently affected genes nor enrichment of specific pathways were observed. Thus, other alterations such as non-coding variants or epigenetic changes might be the major driving force of tumour progression in SPS.

Abstract Background Telomere maintenance mechanisms (TMM) are a hallmark of high-risk neuroblastoma, and are conferred by activation of telomerase or alternative lengthening of telomeres (ALT). However, detection of TMM is not yet part of the clinical routine, and consensus on TMM detection, especially on ALT assessment, remains to be achieved. Methods Whole genome sequencing (WGS) data of 68 primary neuroblastoma samples were analyzed. Telomere length was calculated from WGS data or by telomere restriction fragment analysis (n = 39). ALT was assessed by C-circle assay (CCA, n = 67) and detection of ALT-associated PML nuclear bodies (APB) by combined fluorescence in situ hybridization and immunofluorescence staining (n = 68). RNA sequencing was performed (n = 64) to determine expression of TERT and telomeric long non-coding RNA (TERRA). Telomerase activity was examined by telomerase repeat amplification protocol (TRAP, n = 15). Results Tumors were considered as telomerase-positive if they harbored a TERT rearrangement, MYCN amplification or high TERT expression (45.6%, 31/68), and ALT-positive if they were positive for APB and CCA (19.1%, 13/68). If all these markers were absent, tumors were considered TMM-negative (25.0%, 17/68). According to these criteria, the majority of samples were classified unambiguously (89.7%, 61/68). Assessment of additional ALT-associated parameters clarified the TMM status of the remaining seven cases with high likelihood: ALT-positive tumors had higher TERRA expression, longer telomeres, more telomere insertions, a characteristic pattern of telomere variant repeats, and were associated with ATRX mutations. Conclusions We here propose a workflow to reliably detect TMM in neuroblastoma. We show that unambiguous classification is feasible following a stepwise approach that determines both, activation of telomerase and ALT. The workflow proposed in this study can be used in clinical routine and provides a framework to systematically and reliably determine telomere maintenance mechanisms for risk stratification and treatment allocation of neuroblastoma patients.

Abstract Background Plant development is controlled by the action of many, often connected gene regulatory networks. Differential gene expression controlled by internal and external cues is a major driver of growth and time specific differentiation in plants. Transcriptome analysis is the state-of-the-art method to detect spatio-temporal changes in gene expression during development. Monitoring changes in gene expression at early stages or in small plant organs and tissues requires an accurate technique of tissue isolation, which subsequently results in RNA of sufficient quality and quantity. Laser-microdissection enables such accurate dissection and collection of desired tissue from sectioned material at a microscopic level for RNA extraction and subsequent downstream analyses, such as transcriptome, proteome, genome or miRNA. Results A protocol for laser-microdissection, RNA extraction and RNA-seq was optimized and verified for three distant angiosperm species: Arabidopsis thaliana (Brassicaceae), Oryza sativa (Poaceae) and Eschscholzia californica (Papaveraceae). Previously published protocols were improved in processing speed by reducing the vacuum intensity and incubation time during tissue fixation and incubation time and cryoprotection and by applying adhesive tape. The sample preparation and sectioning of complex and heterogenous flowers produced adequate histological quality and subsequent RNA extraction from micro-dissected gynoecia reliably generated samples of sufficient quality and quantity on all species for RNA-seq. Expression analysis of growth stage specific A. thaliana and O. sativa transcriptomes showed distinct patterns of expression of chromatin remodelers on different time points of gynoecium morphogenesis from the initiation of development to post-meiotic stages. Conclusion Here we describe a protocol for plant tissue preparation, cryoprotection, cryo-sectioning, laser microdissection and RNA sample preparation for Illumina sequencing of complex plant organs from three phyletically distant plant species. We are confident that this approach is widely applicable to other plant species to enable transcriptome analysis with high spatial resolution in non-model plant species. The protocol is rapid, produces high quality sections of complex organs and results in RNA of adequate quality well suited for RNA-seq approaches. We provide detailed description of each stage of sample preparation with the quality and quantity measurements as well as an analysis of generated transcriptomes.

Abstract Background The role of the BARD1 gene in breast cancer (BC) and ovarian cancer (OC) predisposition remains elusive, as published case-control investigations have revealed controversial results. We aimed to assess the role of deleterious BARD1 germline variants in BC/OC predisposition in a sample of 4920 BRCA1/2-negative female BC/OC index patients of the German Consortium for Hereditary Breast and Ovarian Cancer (GC-HBOC). Methods A total of 4469 female index patients with BC, 451 index patients with OC, and 2767 geographically matched female control individuals were screened for loss-of-function (LoF) mutations and potentially damaging rare missense variants in BARD1. All patients met the inclusion criteria of the GC-HBOC for germline testing and reported at least one relative with BC or OC. Additional control datasets (Exome Aggregation Consortium, ExAC; Fabulous Ladies Over Seventy, FLOSSIES) were included for the calculation of odds ratios (ORs). Results We identified LoF variants in 23 of 4469 BC index patients (0.51%) and in 36 of 37,265 control individuals (0.10%), resulting in an OR of 5.35 (95% confidence interval [CI] = 3.17–9.04; P

Abstract Background Germline mutations in the BRIP1 gene have been described as conferring a moderate risk for ovarian cancer (OC), while the role of BRIP1 in breast cancer (BC) pathogenesis remains controversial. Methods To assess the role of deleterious BRIP1 germline mutations in BC/OC predisposition, 6341 well-characterized index patients with BC, 706 index patients with OC, and 2189 geographically matched female controls were screened for loss-of-function (LoF) mutations and potentially damaging missense variants. All index patients met the inclusion criteria of the German Consortium for Hereditary Breast and Ovarian Cancer for germline testing and tested negative for pathogenic BRCA1/2 variants. Results BRIP1 LoF mutations confer a high OC risk in familial index patients (odds ratio (OR) = 20.97, 95% confidence interval (CI) = 12.02–36.57, P